CN1238518C - Grifola frondosa cytoplastic amylose production method - Google Patents
Grifola frondosa cytoplastic amylose production method Download PDFInfo
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Abstract
The present invention relates to an edible medicinal glycoconjugate having the functions of resisting human immunodeficiency viruses, resisting tumors, regulating immune systems, etc., which is prepared from natural fungi by fermentation. The present invention is characterized in that the method comprises: carrying out the liquid submerged fermentation of grifola frondosa strains; obtaining single-component grifola frondosa intracellular polysaccharide from the grifola frondosa mycelia obtained by fermentation through the following steps of cell disruption, hot water leaching, deproteinization or fractional precipitation by ethanol and then deproteinization, decolorization, desalination, column purification, etc. The present invention has the following advantages: (1) since the liquid submerged fermentation is adopted, the edible medicinal glycoconjugate has all advantages of grifola frondosa liquid culture; (2) the grifola frondosa intracellular polysaccharide obtained by the present invention has obvious HIV-resistant efficiency and basically has no toxic or side effect; (3) the grifola frondosa intracellular polysaccharide obtained by the present invention contains a single component, and can be directly prepared into injections or oral medicines for patent medicines.
Description
Technical field
The present invention relates to adopt natural fungi by fermentative preparation a kind of have AIDS virus resisting, antitumor, regulate the edible medicinal glycoconjugate of functions such as immunity system, pass through the liquid submerged fermentation of Grifola frondosa bacterial classification specifically, the maitake mushroom mycelia that obtains fermenting is by cytoclasis, deproteinated, decolouring, desalination, steps such as column purification obtain the Grifola frondosa intracellular polyse of one-component.The Grifola frondosa intracellular polyse that is obtained can directly be prepared into medical injection or edible medicinal oral preparations, have AIDS virus resisting, antitumor, regulate function such as immunity system.
Background technology
Grifola frondosa (Grifola frondosa) has another name called chestnut mushroom, lotus flower bacterium, polyporus frondosus, thousand Buddhist bacterium, the strange fruit of leaf bacterium, cloud gill fungus etc., and the Japanese is called マ イ ケ, and (dance is fine and soft, Maitake).Grifola frondosa is under the jurisdiction of Basidiomycotina on taxonomy, Hymenomycetes, and Aphyllophorales, polyporaceae, tree flower Pseudomonas is a kind of natural rare food and medicament dual-purpose fungi.
Grifola frondosa has like delicious taste and texture as the pheasant
[13], like extremely that in Japan supply falls short of demand.In feudal period, even if Grifola frondosa price height in modern age, even is only just revealed the wild mushroom mysterious place of growth several years to weight and silver equivalence in adopting mushroom person testament.The biologically active substance that it is contained---grifolan has the function of biological response modifier (BRM), is a class ideal immunomodulator, has the laudatory title of " king of mushroom para-immunity ".It has significantly antitumor, anti HIV-1 virus, improve function of immune system, blood sugar regulation, blood fat and cholesterol levels, effect such as hypotensive, generation and transfer, AIDS, hypertension, obesity, diabetes and the immune system dysfunction that prevents cancer cells all there is certain curative effect, and without any side effects.In addition, there is the scholar to think that grifolan is that anti-tumor activity is the strongest in all fungal organism active substances abroad, and oral effective.
The mode of production of Grifola frondosa comprises liquid fermenting and solid fermentation, and traditional solid state fermentation can obtain Grifola Frondosa sporophore, but it is slow to have a mycelium germination, the deficiency that fermentation period is long.Have the following advantages with liquid culture method: (1) is compared with traditional solid state cultivation, has shortened the production cycle greatly; (2) adopt fermentor tank can carry out suitability for industrialized production, improve output greatly; (3) save labour intensity, reduce production costs; (4) constant product quality.
Grifolan can extract from the mycelium of solid state fermentation production and sporophore; Also can extract from liquid submerged fermentation, what wherein extract in the mycelium of fermented liquid is intracellular polyse, and what extract from remaining fermented liquid is exocellular polysaccharide.Because the difference of conditions such as substratum, training method, grifolan also can produce difference at aspects such as its molecular weight, structures.
Acquired immune deficiency syndrome (AIDS) (Acquired Immunodeficiency Syndrome, pathogenic agent AIDS) be human immunodeficiency virus (human Immunodeficiency virus, HIV), clinical manifestation mainly be the condition pathogenic infection and (or) malignant tumour takes place.Acquired immune deficiency syndrome (AIDS) has been propagated rapidly since finding, estimates that according to the World Health Organization when the popular many African countries the earliest of AIDS expect 2010, its average expected life-span will subtract about 30 years longevity because of AIDS.What is more, and the global AIDS popular impetus is also in swift and violent rising, especially South Africa, Latin America and Asia.Still do not have effective methods of treatment at present, case fatality rate is high.
Domestic research to grifolan at present mainly concentrates on its anti-tumor aspect, does not appear in the newspapers as yet about its effect of AIDS virus resisting; And mainly be from Grifola Frondosa sporophore, to extract, the low and composition that comprises a series of molecular weight, Grifola frondosa intracellular polyse that structure is different often that obtains of yield.
Abroad, the more of research is the Nanba professor of Japan, and he has carried out human trial with the grifolan that obtains, and about 80% patient feels well, but hiv virus does not see minimizing; After to Grifola frondosa intracellular polyse sulfation, test, find that the effect of its AIDS virus resisting is enhanced but also increase greatly of toxicity, so can't well deal with problems.The grifolan that he obtained is the composition that comprises a series of molecular weight, Grifola frondosa intracellular polyse that structure is different equally in addition.
Summary of the invention
The objective of the invention is must be not enough in the research both at home and abroad in order to overcome at present, establish a kind of method that obtains a kind of Grifola frondosa intracellular polyse of one-component, it can directly be prepared into have AIDS virus resisting, antitumor, the medical injection or the edible medicinal oral preparations that improve functions such as immunity system.
For achieving the above object, the step that the present invention taked is as follows:
The first step: Grifola frondosa bacterial classification liquid submerged fermentation, its substratum consists of: glucose 1～2%, Semen Maydis powder 0.5～1.5%, cotton seed hull 0.25～0.75%, wheat bran 0.5～1%, potassium primary phosphate 0.05～0.4%, trimagnesium phosphate 0.05～0.4%, calcium chloride 0.02～0.3%, sterilized 30 minutes down at 121 ℃; The liquid submerged fermentation condition is: 22～29 ℃ of leavening temperatures, and ventilating ratio 0.2～1.0vvm, 40～120 rev/mins of stirring velocitys, fermented incubation time 120～168h, inoculum size is 10% (volume ratio).
Second step: the mycelium that the centrifugal back of fermented liquid is obtained carries out cytoclasis and hot water lixiviate.Cytoclasis can adopt tissue to smash method, supersonic method, high pressure crush method to pieces, and cytoclastic purpose is in order to improve the effect of hot water lixiviate intracellular polyse; The condition of hot water lixiviate intracellular polyse is 88～95 ℃ for extracting temperature, and extraction time is 2.5～6 hours, and the ratio of mycelium quality and volume of water is 1: 3～1: 6.
The 3rd step: the alcohol step-by-step precipitation method obtains crude polysaccharide powder, promptly adds raw spirit in the hot water vat liquor, makes that the concentration of volume percent of alcohol reaches 22～38% in the liquid, will get supernatant liquor behind this centrifugal.Add raw spirit again in supernatant liquor, make that the concentration of volume percent of alcohol reaches 55～75% in the liquid, this centrifugal is got 80 ℃ of oven dry down of precipitation after 10 minutes, gained is crude polysaccharide powder.Crude polysaccharide powder promptly can be carried out steps such as deproteinated, decolouring, desalination, column purification after with ultrapure water dissolving (mass percent of Crude polysaccharides is 0.5～2.0% in the solution).
The 4th step: deproteinated.Deproteinated can adopt trichoroacetic acid(TCA) deproteinated method, promptly directly adds trichoroacetic acid(TCA) in the hot water vat liquor, makes that the mass percent concentration of trichoroacetic acid(TCA) reaches 5～25% in the liquid.To promptly can reach Deproteinated purpose behind this centrifugal, the supernatant liquor that is obtained is Crude polysaccharides liquid.Also can adopt Sevag method deproteinated.The step that the Sevag method adopts is: with Crude polysaccharides liquid and chloroform-the propyl carbinol mixed solution (by volume, chloroform: propyl carbinol=mix at 4: 1) mixed in 3: 1 by volume, place tool plug test tube, behind the shake well 30min, centrifugal 1min under 4000 rev/mins condition, then water is separated mutually the water intaking phase with chloroform; Repeat twice of above-mentioned steps again.
The 5th step: decolouring.Decolouring can be adopted the hydrogen peroxide oxidation decolouring, drips 20% hydrogen peroxide solution to faint yellow under the pH8 condition, and 40 ℃ are incubated 1 hour down.
The 6th step: desalination.The method that desalination is adopted is that to place molecular weight cut-off be that it is constant to ionic strength to dialyse with ultrapure water in 10000～12000 the dialysis tubing to the Crude polysaccharides solution with deproteinated and decolouring.
The 7th step: column purification.The method that column purification adopted is to adopt 16/10 DEAE chromatography column, phosphate buffered saline buffer with pH6.0～9.0,1M sodium chloride solution gradient elution, with 1～3ml/min flow velocity wash-out, 5ml/ manages collection, through phenol---sulfuric acid process detects every pipe polysaccharide content by pipe, and Folin-phenol method detects protein content by pipe, and that pipe when peaking simultaneously for the first time with polysaccharide in measuring and protein content is an intracellular polyse required for the present invention with the sample of each three pipe before it and afterwards.These samples are promptly obtained pure Grifola frondosa intracellular polyse after the desalination once more.
Of the present invention third and fourth, five can all implement, also can skip wherein several steps and implement.If do not implement for the 3rd step in general, fourth, fifth step generally will implement so; If implemented for the 3rd step, fourth, fifth step did not generally implement so.
After second step, before the 3rd step, can hot dipping extract or Crude polysaccharides liquid be carried out suitable concentrating according to performance, concentration process carries out in rotatory evaporator, spissated temperature is no more than 60 ℃, and cycles of concentration can be determined as the case may be, but be advisable to be no more than 5 times.
Grifola frondosa bacterial classification of the present invention is available from edible mushrooms technology popularization center, Qingfeng County, Henan Province.
DEAE-Mierocrystalline cellulose of the present invention is a kind of supporting dielectric commonly used in the ion exchange chromatography.
Of the present invention rev/min is rotating speed unit, i.e. the revolution of per minute stirring; Vvm is a ventilation unit, i.e. the volume number of the fermented liquid bubbling air of per minute unit volume; M is a concentration unit, every liter of expression mole; Min is a time unit, the expression minute.
Of the present invention centrifugally under per minute 3000 commentaries on classics conditions, carry out.
The present invention is mass percent if no special instructions.
The Grifola frondosa intracellular polyse that the present invention obtained is the single pure glycoconjugate of component, and wherein protein content is 2%, and monose consists of rhamnosyl, pectinose, seminose, glucose, semi-lactosi, each is formed monose and mainly is connected with the β key, and molecular weight is more than 2,000,000; The glycopeptide mode of connection that has the O-glycosides key, amino acid is formed mainly based on silk amino, glycine and Methionin.
The invention is characterized in the liquid submerged fermentation by the Grifola frondosa bacterial classification, the maitake mushroom mycelia that fermentation is obtained is by cytoclasis, the hot water lixiviate, deproteinated or through deproteinated again after the alcohol fractional precipitation, decolouring, desalination, steps such as column purification obtain the Grifola frondosa intracellular polyse of one-component.
The invention has the advantages that: 1) owing to adopt liquid to give birth to layer fermentation, it has all advantages of Grifola frondosa liquid culture; 2) the Grifola frondosa intracellular polyse that the present invention obtained has the effect of tangible anti-HIV, and has no side effect substantially; 3) the Grifola frondosa intracellular polyse that the present invention obtained is an one-component, can directly be prepared into medical injection or oral medicine.
Description of drawings
Fig. 1 is the Supdex200 gel-filtration collection of illustrative plates of GFP-1.
Fig. 2 is that the polysaccharide of GFP-1 Superdex200 gel-filtration detects collection of illustrative plates.
Fig. 3 is the high-pressure liquid phase collection of illustrative plates of GFP-1.
1. the Superdex200 gel permeation chromatography of GFP-1
Intracellular polyse through the separating obtained GFP-1 component of DEAE-Mierocrystalline cellulose chromatography through Superdex 200HR10/13 post gel permeation chromatography, in 215nm, online detection under the 280nm wavelength, phenol one sulfuric acid process detects polysaccharide content by pipe, and Folin-phenol method detects protein content by pipe.
As seen from Figure 1, GFP-1 is through Superdex 200HR10/13 gel permeation chromatography, and in 215nm, online detection obtains 215nm under the 280nm wavelength, and the overlapping symmetry under the 280nm is unimodal.
As seen from Figure 2, also be polysaccharide by the overlapping unimodal of Figure 11 gained, proteic overlapping single symmetrical elution peak.
2. the high pressure liquid chromatography (HPLC) of GFP-1
As seen from Figure 3, GFP-1 is through the HPLC chromatography, and differential refraction detects and shows that tomographic results is that a sharp-pointed symmetry is unimodal.
Embodiment
Below specific embodiment is described, in the description process of embodiment, is mass percent if no special instructions; Inoculum size is 10% (volume ratio); Centrifugally all under 3000 rpms of conditions, carry out.Concentration process all carries out in rotatory evaporator, and spissated temperature is no more than 60 ℃.The Deproteinated concrete steps of Sevag method described herein are: hot water vat liquor or Crude polysaccharides liquid were mixed with chloroform-propyl carbinol mixed solution in 3: 1 by volume, place tool plug test tube, behind the shake well 30 minutes, under 4000 rev/mins condition centrifugal 1 minute, then water is separated mutually the water intaking phase with chloroform; Repeat above-mentioned steps twice again, wherein, both volume ratios are chloroform in chloroform-propyl carbinol mixed solution: propyl carbinol=4: 1.The decolouring of decolouring employing hydrogen peroxide oxidation then is operating as and drips 20% hydrogen peroxide solution to faint yellow under the pH8 condition, and 40 ℃ are incubated 1 hour down.Dialysis method is all adopted in desalination, and promptly to place molecular weight cut-off be that it is constant to ionic strength to dialyse with ultrapure water in 10000～12000 the dialysis tubing to Crude polysaccharides solution.The condition of column purification is for adopting 16/10DEAE-Mierocrystalline cellulose chromatography post, with phosphate buffered saline buffer, 1M sodium chloride solution gradient elution, with the certain flow rate wash-out, the 5ml/ tube portion is collected, through phenol---sulfuric acid process detects every pipe polysaccharide content by pipe, Folin-phenol method detects protein content by pipe, that pipe when peaking simultaneously for the first time with polysaccharide in measuring and protein content is the intracellular polyse of collection required for the present invention with the sample of each three pipe before it and afterwards, and the intracellular polyse solution of these collections is carried out lyophilize after the desalination once more; Polysaccharide yield is the volume ratio of polysaccharide quality and corresponding fermented liquid.
Embodiment 1: substratum consists of glucose 2%, Semen Maydis powder 1.5%, cotton seed hull 0.75%, wheat bran 1%, potassium primary phosphate 0.4%, trimagnesium phosphate 0.4%, calcium chloride 0.3%, sterilizes 30 minutes down at 121 ℃; 25 ℃ of leavening temperatures, ventilating ratio 0.65vvm, 80 rev/mins of stirring velocitys are cultivated and are finished fermentation after 120 hours; With centrifugal 15 minutes of fermented liquid, collect mycelium; In the ratio of mycelium quality and volume of water is that 1: 5 ratio adds the laggard capable cytoclasis of entry, and hot water lixiviate 6 hours under 90 ℃ of conditions; Be cooled to after the room temperature and got supernatant liquor in centrifugal 15 minutes, concentrate 5 times, to wherein adding trichoroacetic acid(TCA), make that the concentration of trichoroacetic acid(TCA) reaches 10% in the liquid, left standstill the back centrifugal 20 minutes, the gained supernatant liquor is Crude polysaccharides liquid; With the decolouring of Crude polysaccharides liquid, desalination, at the phosphate buffered saline buffer with pH6.0,1M sodium chloride solution gradient elution with column purification under the 1ml/min flow velocity elution requirement, obtains pure Grifola frondosa intracellular polyse GFP-1, and extracting yield is 0.24 ‰.
Embodiment 2: substratum consists of glucose 1.2%, Semen Maydis powder 1.0%, cotton seed hull 0.5%, wheat bran 0.5%, potassium primary phosphate 0.2%, trimagnesium phosphate 0.1%, calcium chloride 0.1%, sterilizes 30 minutes down at 121 ℃; 25 ℃ of leavening temperatures, ventilating ratio 1.0vvm, 40 rev/mins of stirring velocitys are cultivated and are finished fermentation after 168 hours; With centrifugal 15 minutes of fermented liquid, collect mycelium; In the ratio of mycelium quality and volume of water is that 1: 5 ratio adds the laggard capable cytoclasis of entry, and hot water lixiviate 6 hours under 92 ℃ of conditions; Be cooled to after the room temperature and got supernatant liquor in centrifugal 15 minutes, concentrate 3 times, to wherein adding trichoroacetic acid(TCA), make that the concentration of trichoroacetic acid(TCA) reaches 15% in the liquid, left standstill the back centrifugal 20 minutes, the gained supernatant liquor is Crude polysaccharides liquid; With the decolouring of Crude polysaccharides liquid, desalination, at phosphate buffered saline buffer with pH9.0,1M sodium chloride solution gradient elution, with column purification under the 3ml/min flow velocity elution requirement, the back obtains pure Grifola frondosa intracellular polyse GFP-1, and extracting yield is 0.28 ‰.
Embodiment 3: substratum consists of glucose 1.8%, Semen Maydis powder 1.0%, cotton seed hull 0.5%, wheat bran 0.8%, potassium primary phosphate 0.2%, trimagnesium phosphate 0.2%, calcium chloride 0.05%, sterilizes 30 minutes down at 121 ℃; 29 ℃ of leavening temperatures, ventilating ratio 0.2vvm, 120 rev/mins of stirring velocitys are cultivated and are finished fermentation after 132 hours; With centrifugal 15 minutes of fermented liquid, collect mycelium; In the ratio of mycelium quality and volume of water is that 1: 3 ratio adds the laggard capable cytoclasis of entry, and hot water lixiviate 2.5 hours under 95 ℃ of conditions; Be cooled to after the room temperature and got supernatant liquor in centrifugal 15 minutes, to wherein adding trichoroacetic acid(TCA), make that the concentration of trichoroacetic acid(TCA) reaches 25% in the liquid, left standstill the back centrifugal 20 minutes, the gained supernatant liquor is Crude polysaccharides liquid; With the decolouring of Crude polysaccharides liquid, desalination, at phosphate buffered saline buffer with pH6.0,1M sodium chloride solution gradient elution, with column purification under the 3ml/min flow velocity elution requirement, the back obtains pure Grifola frondosa intracellular polyse GFP-1, and extracting yield is 0.24 ‰.
Embodiment 4: substratum consists of glucose 1%, Semen Maydis powder 0.5%, cotton seed hull 0.25%, wheat bran 0.5%, potassium primary phosphate 0.05%, trimagnesium phosphate 0.05%, calcium chloride 0.02%, sterilizes 30 minutes down at 121 ℃; 25 ℃ of leavening temperatures, ventilating ratio 0.40vvm, 100 rev/mins of stirring velocitys are cultivated and are finished fermentation after 120 hours; With centrifugal 15 minutes of fermented liquid, collect mycelium; In the ratio of mycelium quality and volume of water is that 1: 5 ratio adds the laggard capable cytoclasis of entry, and hot water lixiviate 6 hours under 90 ℃ of conditions; Be cooled to after the room temperature and got supernatant liquor in centrifugal 15 minutes, to wherein adding trichoroacetic acid(TCA), make that the concentration of trichoroacetic acid(TCA) reaches 5% in the liquid, left standstill the back centrifugal 20 minutes, the gained supernatant liquor is Crude polysaccharides liquid; With the decolouring of Crude polysaccharides liquid, desalination, at phosphate buffered saline buffer with pH9.0,1M sodium chloride solution gradient elution, with column purification under the 1ml/min flow velocity elution requirement, the back obtains pure Grifola frondosa intracellular polyse GFP-1, and extracting yield is 0.26 ‰.
Embodiment 5: substratum consists of glucose 1%, Semen Maydis powder 1.5%, cotton seed hull 0.25%, wheat bran 0.75%, potassium primary phosphate 0.3%, trimagnesium phosphate 0.1%, calcium chloride 0.1%, sterilizes 30 minutes down at 121 ℃; 22 ℃ of leavening temperatures, ventilating ratio 0.65vvm, 80 rev/mins of stirring velocitys are cultivated and are finished fermentation after 168 hours; With centrifugal 15 minutes of fermented liquid, collect mycelium; In the ratio of mycelium quality and volume of water is that 1: 6 ratio adds the laggard capable cytoclasis of entry, and hot water lixiviate 4.5 hours under 92.5 ℃ of conditions; Be cooled to after the room temperature and got supernatant liquor in centrifugal 15 minutes, concentrate 5 times, add raw spirit, make that the concentration of volume percent of alcohol reaches 30% in the liquid, this centrifugal was got supernatant liquor after 10 minutes.Add raw spirit again in supernatant liquor, make that the concentration of volume percent of alcohol reaches 60% in the liquid, this centrifugal is got 80 ℃ of oven dry down of precipitation after 10 minutes, gained is crude polysaccharide powder.Crude polysaccharide powder is dissolved into Crude polysaccharides solution with ultrapure water, and the mass percent of Crude polysaccharides is 0.5% in the solution.With this Crude polysaccharides liquid desalination, at phosphate buffered saline buffer with pH6.8,1M sodium chloride solution gradient elution, with column purification under the 1ml/min flow velocity elution requirement, the back obtains pure Grifola frondosa intracellular polyse GFP-1, and extracting yield is 0.25 ‰.
Embodiment 6: substratum consists of glucose 1.2%, Semen Maydis powder 1.0%, cotton seed hull 0.5%, wheat bran 0.5%, potassium primary phosphate 0.2%, trimagnesium phosphate 0.1%, calcium chloride 0.1%, sterilizes 30 minutes down at 121 ℃; 25 ℃ of leavening temperatures, ventilating ratio 0.45vvm, 120 rev/mins of stirring velocitys are cultivated and are finished fermentation after 120 hours; With centrifugal 15 minutes of fermented liquid, collect mycelium; In the ratio of mycelium quality and volume of water is that 1: 4 ratio adds the laggard capable cytoclasis of entry, and hot water lixiviate 3.5 hours under 88 ℃ of conditions; Be cooled to after the room temperature and got supernatant liquor in centrifugal 15 minutes, concentrate 2 times, add raw spirit, make that the concentration of volume percent of alcohol reaches 22% in the liquid, this centrifugal was got supernatant liquor after 10 minutes.Add raw spirit again in supernatant liquor, make that the concentration of volume percent of alcohol reaches 55% in the liquid, this centrifugal is got 80 ℃ of oven dry down of precipitation after 10 minutes, gained is crude polysaccharide powder.Crude polysaccharide powder is dissolved into Crude polysaccharides solution with ultrapure water, and the mass percent of Crude polysaccharides is 2.0% in the solution.With desalination again behind this Crude polysaccharides liquid usefulness sevag method deproteinated, at phosphate buffered saline buffer with pH6.8,1M sodium chloride solution gradient elution, with column purification under the 2ml/min flow velocity elution requirement, the back obtains pure Grifola frondosa intracellular polyse GFP-1, and extracting yield is 0.24 ‰.
Embodiment 7: substratum consists of glucose 1.8%, Semen Maydis powder 1.0%, cotton seed hull 0.5%, wheat bran 0.8%, potassium primary phosphate 0.2%, trimagnesium phosphate 0.2%, calcium chloride 0.05%, sterilizes 30 minutes down at 121 ℃; 25 ℃ of leavening temperatures, ventilating ratio 0.8vvm, 40 rev/mins of stirring velocitys are cultivated and are finished fermentation after 136 hours; With centrifugal 15 minutes of fermented liquid, collect mycelium; In the ratio of mycelium quality and volume of water is that 1: 3.5 ratio adds the laggard capable cytoclasis of entry, and hot water lixiviate 2.5 hours under 95 ℃ of conditions; Be cooled to after the room temperature and got supernatant liquor in centrifugal 15 minutes, add raw spirit, make that the concentration of volume percent of alcohol reaches 38% in the liquid, this centrifugal was got supernatant liquor after 10 minutes.Add raw spirit again in supernatant liquor, make that the concentration of volume percent of alcohol reaches 75% in the liquid, this centrifugal is got 80 ℃ of oven dry down of precipitation after 10 minutes, gained is crude polysaccharide powder.Crude polysaccharide powder is dissolved into Crude polysaccharides solution with ultrapure water, and the mass percent of Crude polysaccharides is 1.5% in the solution.With this Crude polysaccharides liquid decolouring, desalination, at phosphate buffered saline buffer with pH7.8,1M sodium chloride solution gradient elution, with column purification under the 1.5ml/min flow velocity elution requirement, the back obtains pure Grifola frondosa intracellular polyse GFP-1, and extracting yield is 0.26 ‰.
Embodiment 8: substratum consists of glucose 1.2%, Semen Maydis powder 1.0%, cotton seed hull 0.5%, wheat bran 0.5%, potassium primary phosphate 0.2%, trimagnesium phosphate 0.1%, calcium chloride 0.1%, sterilizes 30 minutes down at 121 ℃; 22 ℃ of leavening temperatures, ventilating ratio 1.0vvm, 60 rev/mins of stirring velocitys are cultivated and are finished fermentation after 148 hours; With centrifugal 15 minutes of fermented liquid, collect mycelium; In the ratio of mycelium quality and volume of water is that 1: 6 ratio adds the laggard capable cytoclasis of entry, and hot water lixiviate 6 hours under 90 ℃ of conditions; Be cooled to after the room temperature and got supernatant liquor in centrifugal 15 minutes, add raw spirit, make that the concentration of volume percent of alcohol reaches 32% in the liquid, this centrifugal was got supernatant liquor after 10 minutes.Add raw spirit again in supernatant liquor, make that the concentration of volume percent of alcohol reaches 68% in the liquid, this centrifugal is got 80 ℃ of oven dry down of precipitation after 10 minutes, gained is crude polysaccharide powder.Crude polysaccharide powder is dissolved into Crude polysaccharides solution with ultrapure water, and the mass percent of Crude polysaccharides is 2.0% in the solution.To in this Crude polysaccharides liquid, add trichoroacetic acid(TCA), make that the concentration of trichoroacetic acid(TCA) reaches 10% in the liquid, left standstill the back centrifugal 20 minutes, the gained supernatant liquor decolours again, desalination, at the phosphate buffered saline buffer with pH6.8,1M sodium chloride solution gradient elution is with column purification under the 1ml/min flow velocity elution requirement, the back obtains pure Grifola frondosa intracellular polyse GFP-1, and extracting yield is 0.23 ‰.
Embodiment 9: substratum consists of glucose 1.8%, Semen Maydis powder 1.0%, cotton seed hull 0.5%, wheat bran 0.8%, potassium primary phosphate 0.2%, trimagnesium phosphate 0.2%, calcium chloride 0.05%, sterilizes 30 minutes down at 121 ℃; 28 ℃ of leavening temperatures, ventilating ratio 0.65vvm, 100 rev/mins of stirring velocitys are cultivated and are finished fermentation after 140 hours; With centrifugal 15 minutes of fermented liquid, collect mycelium; In the ratio of mycelium quality and volume of water is that 1: 4 ratio adds the laggard capable cytoclasis of entry, and hot water lixiviate 5 hours under 88 ℃ of conditions; Be cooled to after the room temperature and got supernatant liquor in centrifugal 15 minutes, concentrate 1 times, add raw spirit, make that the concentration of volume percent of alcohol reaches 26% in the liquid, this centrifugal was got supernatant liquor after 10 minutes.Add raw spirit again in supernatant liquor, make that the concentration of volume percent of alcohol reaches 70% in the liquid, this centrifugal is got 80 ℃ of oven dry down of precipitation after 10 minutes, gained is crude polysaccharide powder.Crude polysaccharide powder is dissolved into Crude polysaccharides solution with ultrapure water, and the mass percent of Crude polysaccharides is 1.75% in the solution.With this Crude polysaccharides liquid desalination, at phosphate buffered saline buffer with pH6.8,1M sodium chloride solution gradient elution, with column purification under the 1.5ml/min flow velocity elution requirement, the back obtains pure Grifola frondosa intracellular polyse GFP-1, and extracting yield is 0.28 ‰.
Claims (12)
1, a kind of method of production Grifola frondosa intracellular polyse, it is characterized in that: a, submerged fermentation, with Grifola frondosa liquid at 22～29 ℃ of leavening temperatures, ventilating ratio 0.2～1.0vvm, 40～120 rev/mins of stirring velocitys, submerged fermentation under the condition of fermented incubation time 120～168h; B, cytoclasis are carried out fragmentation with the mycelium in the above-mentioned fermented liquid; C, hot water lixiviate are 88～95 ℃ in temperature, and the time is 2.5～6 hours, and the ratio of mycelium quality and volume of water is to carry out lixiviate under 1: 3～1: 6 the condition, obtains containing the vat liquor of intracellular polyse; D, desalination utilize dialysis method that Crude polysaccharides solution is carried out desalting treatment; E, column purification utilize the 16/10DEAE chromatography column that the Crude polysaccharides solution that takes off supersalt is carried out column purification; The used substratum of Grifola frondosa liquid submerged fermentation consists of: glucose 1～2%, Semen Maydis powder 0.5～1.5%, cotton seed hull 0.25～0.75%, wheat bran 0.5～1%, potassium primary phosphate 0.05～0.4%, trimagnesium phosphate 0.05～0.4%, calcium chloride 0.02～0.3%.
2, by the method for the described production Grifola frondosa of claim 1 intracellular polyse, it is characterized in that: when cytoclasis, will smash method or supersonic method or high pressure crush method to pieces through the mycelium utilization tissue after the submerged fermentation and carry out cytoclasis.
3, method by the described production Grifola frondosa of claim 1 intracellular polyse, it is characterized in that: after the hot water lixiviate, before the desalination, utilize alcohol to above-mentioned vat liquor fractional precipitation, promptly in the hot water vat liquor, add raw spirit, make that the concentration of volume percent of alcohol reaches 22～38% in the liquid, to get supernatant liquor behind this centrifugal, in supernatant liquor, add raw spirit again, make that the concentration of volume percent of alcohol reaches 55～75% in the liquid, in 80 ℃ of oven dry down, gained is crude polysaccharide powder with taking precipitate behind this centrifugal, crude polysaccharide powder is dissolved into Crude polysaccharides solution with ultrapure water, and the mass percent of Crude polysaccharides is 0.5～2.0% in the solution.
4, by the method for the described production Grifola frondosa of claim 1 intracellular polyse, it is characterized in that: after the hot water lixiviate, deproteinated again.
5, by the method for the described production Grifola frondosa of claim 5 intracellular polyse, it is characterized in that: behind deproteinated, decolouring again, promptly in above-mentioned Crude polysaccharides solution, drip 20% hydrogen peroxide solution to faint yellow, when dripping 20% hydrogen peroxide solution, the pH value of Crude polysaccharides is 8, is incubated 1 hour down at 40 ℃ again.
6, by the method for the described production Grifola frondosa of claim 1 intracellular polyse, it is characterized in that: when desalination, it is that it is constant to ionic strength to dialyse with ultrapure water in 10000～12000 the dialysis tubing that the Crude polysaccharides solution that has decoloured is placed molecular weight cut-off.
7, press the method for the described production Grifola frondosa of claim 1 intracellular polyse, it is characterized in that: when column purification, adopt the 16/10DEAE chromatography column, phosphate buffered saline buffer with pH6.8～9.0,1M sodium chloride solution gradient elution, with 1～3ml/min flow velocity wash-out, the 5ml/ tube portion is collected, through phenol---sulfuric acid process detects every pipe polysaccharide content by pipe, Folin-phenol method detects protein content by pipe, and that pipe when peaking simultaneously for the first time with polysaccharide in measuring and protein content is required intracellular polyse with the sample of each three pipe before it and afterwards.
8, by the method for the described production Grifola frondosa of claim 7 intracellular polyse, it is characterized in that: the Grifola frondosa intracellular polyse that is obtained is formed monose and mainly is connected with the β key for each, and molecular weight is at the single pure glycoconjugate of 2,000,000 above components.
9, by the method for the described production Grifola frondosa of claim 3 intracellular polyse, it is characterized in that: after obtaining Crude polysaccharides solution, before the desalination, carry out deproteinated.
10, press the method for claim 3 or 9 described production Grifola frondosa intracellular polyses, it is characterized in that: after obtaining Crude polysaccharides solution or deproteinated, decolouring again, promptly in above-mentioned Crude polysaccharides solution, drip 20% hydrogen peroxide solution to faint yellow, when dripping 20% hydrogen peroxide solution, the pH value of Crude polysaccharides is 8, is incubated 1 hour down at 40 ℃ again.
11, press the method for claim 4 or 9 described production Grifola frondosa intracellular polyses, it is characterized in that: when deproteinated, in hot water vat liquor or Crude polysaccharides solution, directly add trichoroacetic acid(TCA), make that the mass percent concentration of trichoroacetic acid(TCA) reaches 5～25% in the liquid, again with this centrifugal, slough albumen wherein, the supernatant liquor that is obtained is Crude polysaccharides solution.
12, press the method for claim 4 or 9 described production Grifola frondosa intracellular polyses, it is characterized in that: when deproteinated, in hot water vat liquor or Crude polysaccharides solution, utilize Sevag method deproteinated: hot water vat liquor or Crude polysaccharides liquid were mixed with chloroform-propyl carbinol mixed solution in 3: 1 by volume, place tool plug test tube, behind the shake well 30 minutes, under 4000 rev/mins condition centrifugal 1 minute, then water is separated mutually the water intaking phase with chloroform; Repeat above-mentioned steps twice again, wherein, both volume ratios are chloroform in chloroform-propyl carbinol mixed solution: propyl carbinol=4: 1.
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| CN 200310106540 CN1238518C (en) | 2003-12-03 | 2003-12-03 | Grifola frondosa cytoplastic amylose production method |
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